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Food Control 19 (2008) 1183–1190

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Comparative study of DNA extraction methods for soybean

derived food products
Isabel Mafra *, Susana A. Silva, Elsa J.M.O. Moreira, Carla S. Ferreira da Silva,
M. Beatriz, P.P. Oliveira
REQUIMTE – Servicßo de Bromatologia, Faculdade de Farmácia, Universidade do Porto, Rua Anı́bal Cunha, 164, 4099-030 Porto, Portugal

Received 24 August 2007; received in revised form 7 January 2008; accepted 15 January 2008

Abstract

The present work describes the comparison of four DNA extraction methods applied to a wide range of soybean derived food prod-
ucts. The methods included the commercial kits NucleoSpin and GeneSpin, the CTAB, and the Wizard methods. The protocols have
been compared for their extraction efficiency, evaluated by the determination of yield and purity of DNA extracts, as well as amplifiabil-
ity. All the methods produced DNA suitable for PCR amplification for the majority of analysed foods, with the exception of soybean
sauces and some processed foods. The NucleoSpin and GeneSpin methods showed the best results for DNA yield and purity, when
applied to soybean flours and protein isolates. The CTAB and Wizard methods were generally well suited to all kind the food matrices
tested. The extraction of processed foods, such as drinks, desserts or vegetarian foods was better achieved with the CTAB or the Wizard
methods as both produced high number of amplifiable extracts. The four methods showed similar performances for real-time PCR
amplification.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: DNA extraction; Soybean; Food products; PCR; GMO detection

1. Introduction The need to monitor and verify the presence and

The soybean (Glycine max) is the most important genet-
ically modified crop, from which 60% of the word’s planted
area corresponds to the Roundup Ready (RR) soybeans
(James, 2006). Since the genetically modified organisms
(GMOs) entered the food chain, a scientific and public
debate concerning their safety and the need for labelling
information came up especially in Europe. For this reason,
the EU has dedicated special attention to consumer infor-
mation by requiring a compulsory labelling for food prod-
ucts containing more than 0.9% of authorised GM material
(Regulation (EC) No. 1829/2003, 2003).
*
Corresponding author. Tel.: +351 222078902; fax: +351 222003977.
E-mail address: isabel.mafra@ff.up.pt (I. Mafra).
amounts of biotechnology derived material in food
products demands analytical methods able to detect, to
identify and to quantify either the introduced DNA or
the expressed protein(s). The DNA based methods, namely
PCR techniques, are the methods of choice due to their
high sensitivity and specificity, which allow the detection
of very small amounts of DNA in raw materials and pro-
cessed foods (Engel, Moreano, Ehlert, & Busch, 2006;
Holst-Jensen, Rønning, Løvseth, & Berdal, 2003). So, the
DNA amplification methods are emerging as useful tech-
niques in food inspection and regulation, not only in the
detection of GMOs in foodstuffs but also in the detection
of food adulterations (Calvo, Osta, & Zaragoza, 2002;
Comi, Iacumin, Rantsiou, Cantoni, & Cocolin, 2004;
Mafra, Ferreira, Faria, & Oliveira, 2004; Poms, Klein, &
Anklam, 2004; Woolfe & Primrose, 2004).

0956-7135/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2008.01.004
1184 I. Mafra et al. / Food Control 19 (2008) 1183–1190
However, the successful of DNA amplification methods
depends on the efficient DNA extraction protocols, which
should provide high quantity and quality DNA. The food
products are complex matrices that might contain a num-
ber of PCR inhibitors such as polysaccharides, polyphenols
and proteins (Terry, Harris, & Parkers, 2002). Further-
more, they have frequently been subjected to one or more
steps of processing, such as mechanical, thermal, chemical
or enzymatic treatment, affecting the integrity of DNA.
Consequently, the isolation of DNA from foods can some-
times be a challenge for food analysis. Considering the high
range of soybean products supplied by the food industry,
the variety of food matrices with different types of process-
ing, and the great number of protocols available for DNA
extraction, to know the best method(s) for each type of
matrix imply many combinations of analysis.
Several authors have compared many protocols for
DNA extraction of foods. However, the range of products
analysed is limited in most reports or include only one rep-
resentative of each type of food. Zimmerman, Lüthy, and
Pauli (1998) compared nine different extraction methods
from soybean food samples verifying that five of them
degraded the extracted DNA while the other four (CTAB,
Wizard, DNeasy and Nucleon Phytopure) gave relatively
low yields but good quality DNA. However, the number
of samples analysed was small including only tofu, soybean
flour and soybean lecithin. Other authors have verified
good performances for the CTAB method for soybean
DNA in chocolate and biscuits together with other DNA
binding kits (Gryson, Messens, & Dewettinck, 2004). Hol-
den et al. (2003) verified that the methods employing bind-
ing DNA to a solid matrix (silica gel or magnetic particles)
performed better for PCR amplifications than the selective
precipitation methods, with the exception of CTAB, when
applied to grounded corn. On the other hand, Smith and
Maxwell (2007) verified that the Wizard method was the
most efficient method for the corn flour and three cornst-
archs, when compared to CTAB method and binding
DNA to solid matrices, including magnetic particles. Ole-
xová, Dovičovičová, and Kuchta (2004) observed good
performances of DNA extraction by using the CTAB and
the binding DNA to a solid matrix with commercial kits
applied to soybean, maize and wheat products (flours, bis-
cuits and instant pap).
In the present work, we have compared the performance
of four DNA extraction methods in several soybean
derived food products. Taking into consideration the suc-
cessful results reported for DNA extraction from different
food matrices, we chose the in house developed CTAB
method, the partial use of the commercial kit WizardÒ
and the commercial kits NucleoSpin and GeneSpin, for
their simplicity. The food samples analysed included a wide
range of soybean products, with different degrees of pro-
cessing and several representatives of each type of food,
namely: grounded soybean, protein isolates, soybean
drinks, tofu, sauces, desserts and precooked vegetarian
foods, obtained from local supermarkets.
2. Materials and methods
2.1. Samples
The samples included raw soybeans supplied by a food
industry (3 samples) and purchased in a local market (2
samples) and processed products: soybean flour (1), protein
isolates (7), soybean drinks (8, including 1 powder drink),
soybean sauces (4), tofu (5), soybean desserts (6) and sev-
eral precooked vegetarian food products containing soy-
bean or tofu (soybean hamburgers, soybean sausages and
other soybean and/or tofu containing meals). The pro-
cessed products were purchased in local grocery retailers
and supermarkets.
2.2. DNA extraction
The samples were extracted with four different meth-
ods: NucleoSpin, GeneSpin, CTAB and Wizard. Two
methods consisted on the use of the commercial kits Nucle-
oSpin (Macherey-Nagel, Düren, Germany) and GeneSpin
(GeneScan, Freiburg, Germany) according to the manu-
facture instructions, which were equivalent, changing the
lysis incubation time from 30 to 60 min at 65 °C in both
kits.
CTAB method. The CTAB (cetyltrimethylammonium
bromide) method was performed as described by Lipp,
Brodmann, Pietsch, Pauwels, and Anklam (1999) with
some modifications. The samples (100–500 mg of grounded
and homogenised sample) were transferred to a 2 mL ster-
ile reaction tube followed by the addition of 1 mL of CTAB
extraction buffer (20 g CTAB/L, 1.4 M NaCl, 0.1 M Tris–
HCl, 20 mM EDTA) and mixed. After the incubation at
65 °C for 1 h, with occasional stirring, the suspension was
centrifuged (15 min, 18,514g) and 500 lL of the superna-
tant were extracted with 400 lL of chloroform, stirred
and centrifuged (10 min, 12,000g). The upper phase was
transferred to a new tube, mixed with double volume of
CTAB precipitation solution (5 g/L, 0.04 M NaCl) and
incubated for 1 h at room temperature. After centrifuga-
tion (10 min, 12,000g), the supernatant was discarded and
the precipitate was dissolved in 350 lL of 1.2 M NaCl
and extracted with 350 lL chloroform. The mixture was
centrifuged (10 min, 12,000g) and the upper phase was
mixed with 0.6 volume parts of isopropanol at 20 °C.
The mixture was centrifuged (10 min, 12,000g), the super-
natant was discarded and the pellet was washed with
500 lL of ethanol solution (70% v/v) at 20 °C. After
centrifugation, the supernatant was discarded carefully
by pipeting, the pellet was dried and the DNA was dis-
solved in 100 lL of Tris–EDTA buffer (10 mM Tris,
1 mM EDTA).
Wizard method. The Wizard method consisted on the
use of the columns and WizardÒ resin (Promega, Madison,
WI, USA) as described by Lipp et al. (1999) with some
modifications. The samples (100–500 mg of grounded
and homogenised sample) were transferred to a 2 mL
 I. Mafra et al. / Food Control 19 (2008) 1183–1190
sterile reaction tube followed by the addition of 860 lL of
extraction buffer TNE (10 mM Tris, 150 mM NaCl, 2 mM
EDTA, 1% SDS), 100 lL of 5 M guanidine hydrochloride
solution and 40 lL proteinase K solution (20 mg/mL).
After the incubation at 60 °C for 3 h, with occasional
stirring, the suspension was centrifuged (15 min, 18,514g)
and 500 lL of the supernatant were mixed with 1 mL of
WizardÒ DNA purification resin (Promega, Madison,
WI, USA). A 2 mL syringe was mounted on the column
and the mixture was pushed with the plunger through the
column. The DNA-resin mixture was washed with 2 mL
isopropanol solution (80% v/v). The column was dried
for 5 min at room temperature and mounted on a new
reaction tube. The DNA was eluted by the addition of
100 lL of Tris–EDTA buffer (10 mM Tris, 1 mM EDTA)
at 70 °C, incubation for 1 min and centrifugation (1 min,
10,000g).
The extractions were done in duplicate assays for each
sample. The quality of extracted DNA was analysed by
electrophoresis in a 1.0% agarose gel carried out in TAE
buffer (40 mM Tris–acetate, 1 mM EDTA) for 40 min at
120 V, stained with ethidium bromide (0.4 lg mL 1 for
5 min) and destained in distilled water for 20 min. The aga-
rose gel was visualised under UV light and a digital image
was obtained using a Kodak Digital Science DC120 TM
(Rochester, NY, USA).
2.3. DNA quantification and purity
The DNA was quantified using the Picogreen reagent
(Molecular Probes) according to the kit instructions. k
DNA supplied by the kit was used for preparing standard
curves ranging from 1.0 to 1000 ng/mL, and sample
extracts were diluted to 1/100. Both standard and samples
were diluted in TE 1 supplied by the kit. The fluorescence
was measured in a spectrofluorophotometer Shimadzu RF-
540 (Shimadzu Corporation, Kyoto, Japan) using an excit-
atory wavelength of 480 nm at an emission wavelength of
520 nm.
The purity of the extracted DNA was determined by the
ratio of the absorbance at 260 and 280 nm using a spectro-
photometer Shimadzu UV-160A (Shimadzu Corporation,
Kyoto, Japan).
2.4. Oligonucleotide primers
The oligonucleotide primer pairs used were: LE1 (CCA
AGC AAT GGC TAC TTC AAA G), LE2 (TGA GTT
TGC CTT GCT GGT CAG T), LE3 (GCA AAG CAA
TGG CTA CTT CAA) and LE4 (AAG AAA CAG TTT
CCG CTG AGT T). The primers were designed based on
the lectin gene (Genbank Accession No. K00821) by a
software (http://seq.yeastgenome.org) and the nucleotide
sequences were submitted to a BLASTn sequence similarity
search, which confirmed primer specificity. The primers
were synthesised by MWG-BIOTECH AG (Ebersberg,
Germany).
1185
2.5. PCR amplification
The amplifications by polymerase chain reaction (PCR)
were carried out in 25 lL total reaction volume containing
2 lL of DNA (50–200 ng) extract, 15 mM Tris–HCl (pH
8.3), 50 mM KCl, 0.5 lM of each primer (LE3 and LE4),
0.2 mM of each dNTP (Invitrogene, Carlsbad, CA,
USA), 2 mM MgCl2 and 1 U of DNA polymerase AmpliT-
aq GoldÒ (Applied Biosystems, Branchburg, NJ, USA).
The PCR amplifications were performed in a thermal
cycler PTC-100 (MJ Research, Inc., Watertown, MA,
USA) using the following program: initial denaturation at
94 °C for 4 min, 35 cycles of 30 s at 94 °C, 30 s at 60 °C
and 30 s at 72 °C, and a final extension at 72 °C for 4 min.
The amplified fragments were analysed by electrophore-
sis in a 2.0% agarose gel carried out in TAE buffer (40 mM
Tris–acetate, 1 mM EDTA) for 60 min at 120 V, stained
with ethidium bromide (0.4 lg mL 1 for 5 min) and
destained in distilled water for 30 min. The agarose gel
was visualised under UV light and a digital image was
obtained using a Kodak Digital Science DC120 (Roches-
TM
ter, NY, USA).
The limit of detection was determined by 10-fold serial
dilutions of the extracts, where the lowest concentration
of DNA that produced a visible PCR product with the
expected size was assigned as the detection limit.
For control of PCR inhibition the spiking test was per-
formed by the addition of 1 lL containing 50 ng of tem-
plate DNA to the PCR mix.
Each extract was amplified at least in duplicate assays.
2.6. Amplification by real-time PCR
The amplification by real-time PCR was carried out in
20 lL containing 2 lL of DNA extract, 1 iQTM SYBRÒ
Green Mix (Bio-Rad Laboratories, Hercules, CA, USA)
and 0.5 mM of each primer (LE1 and LE2).
The real-time PCR assays were performed on a fluoro-
metric thermal cycler iCycler iQTM Real-time Detection Sys-
tem (Bio-Rad Laboratories, Hercules, CA, USA) using the
following conditions: 94 °C for 4 min; 45 cycles at 94 °C for
30 s and 65 °C for 1 min, with collection of fluorescence
signal at the end of each cycle; 60 cycles at 65 °C for 10 s
with increase setpoint temperature after cycle 2 by 0.5 °C
and melt curve data collection. Data was collected and pro-
cessed using a iCycler iQTM Real-Time Detection System
Software version 3.0 (Bio-Rad Laboratories, Hercules,
CA, USA). Each dilution was amplified in duplicate.
2.7. Statistical analysis
The Student’s t test was used to measure the significance
of differences between DNA yields obtained with Nucleo-
Spin and GeneSpin kits. The one-way ANOVA was per-
formed to evaluate differences among real-time PCR
efficiencies obtained with the four methods of extraction.
1186 I. Mafra et al. / Food Control 19 (2008) 1183–1190

SPSS for Windows version 14.0 (SPSS Inc., Chicago, IL,
USA) was used for statistical treatment of results.
3. Results
3.1. Qualitative and quantitative analysis of the extracted
DNA
In the present work, the DNA was isolated from soy-
bean derived foodstuffs using four DNA extraction meth-
ods: the commercial kits NucleoSpin Food Kit
(Macherey-Nagel, Düren, Germany) and GeneSpin (Gene-
Scan, Freiburg, Germany), the CTAB, and the Wizard
resin and columns (Promega, Madison, WI, USA).
The results of DNA yield estimation by fluorescence for
the wide range of products analysed are condensed in
Fig. 1a and 1b for the foods with high and low DNA yields,
a 200
180 Soybean flour
160
Protein isolates
Tofu
140
ng DNA/ mg
respectively. Soybean flours, protein isolates and tofu were
the products with the highest DNA yields by using the four
methods of extraction (>20 ng/mL). The NucleoSpin and
GeneSpin kits enabled the highest yields for the six differ-
ent samples of soybean flours and for the seven different
samples of protein isolates (Fig. 1a and Table 1). The
CTAB method showed considerable lower DNA amounts
than the kits for the soybean flours, protein isolates and
tofu. The Wizard method allowed proximate yields for
the tofu, but much lower DNA yields for soybean flours
and protein isolates than the kits. Although the DNA
yields differed generally with the type of extraction method,
the purity of the extracts obtained from soybean flours,
protein isolates and tofu was high, regardless the extraction
method (Table 1) (A260/A280 > 1.6, for most extracts).
The Student’s t test was used to measure the significance
of differences between paired samples extracted with both
kits NucleoSpin and GeneSpin. As the protocols of both
kits are identical, we wanted to know whether the different
suppliers introduced some difference in reagent composi-
tion (not available) that might change the final yield of
DNA extracts. The results show no significant differences
between DNA yields obtained with the two kits as for soy-
bean flours, protein isolates and tofu, P was higher than
0.05.
Considering the extraction of soybean drinks, the pow-

120 der drink sample was the only successfully extracted by

100
all the methods (Table 1). The other ready to drink samples
showed very reduced or no DNA yield using the kits, but
80 higher yields with the CTAB and Wizard methods (Table
60 1, Fig. 1b). The purity of DNA extracts of soybean drinks
40
was high, as the average ratio A260/A280 was 1.7.
The extraction of soybean sauces resulted in low quality
20 DNA extracts using all the protocols, as the yields were
0 null or very low and the average DNA purity was lower
Nucleospin Genespin CTAB Wizard than 1.6 (A260/A280 = 1.5) (Table 1).
Extraction method The extraction of several highly processed vegetarian
foods and soybean desserts shows a high dispersion of
b 30
DNA yields (Table 1, Fig. 1b). The variability of product
Drinks composition and processing included in each one of these
25 two groups should be responsible for the differences
Desserts
Vegetarian foods observed. Consequently, it was obtained a wide range of
20 DNA yields, as noted in the error bars concerning the stan-
ng DNA/ mg

dard deviate from different food samples. The Wizard

15 method allowed the higher average yields for both vegetar-
ian foods and desserts (Fig. 1b). The purity of dessert
extracts was the best when using the CTAB method (aver-
10
age ratio A260/A280 = 1.8), although this was the method
with lowest DNA yields (Table 1). The NucleoSpin and
5 GeneSpin kits produced similar or slightly higher DNA
yields than the CTAB for the desserts, but the purity was
0 quite low for one of the dessert samples (A260/A280 =
Nucleospin Genespin CTAB Wizard 1.0–1.1) extracted with both kits (Table 1). The purity
Extraction method range of vegetarian food extracts did not change much with
Fig. 1. Average amount of DNA obtained per mg of soybean derived the method of extraction.
food product. The error bars represent the standard deviation around the The extracts were tested for DNA degradation by deter-
mean values of each group products analysed at least in duplicate assays. mining the range of fragment size of the DNA isolated by
 I. Mafra et al. / Food Control 19 (2008) 1183–1190 1187

Table 1
Comparison of DNA yield, purity, PCR and detection limits of PCR positive amplification for several soybean foodstuffs extracted with different methods
Sample Extraction method DNA (ng/lL extract) A260/A280 PCR (+/total) Detection limit (ng)
Soybean flours (100 mg) Nucleospin 139.0–161.4 1.5–2.0 6/6 0.279–0.323
Genespin 146.2–182.2 1.7–2.0 6/6 0.306–0.364
CTAB 19.9–33.9 1.7–2.0 6/6 0.398–0.678
Wizard 86.0–155.4 1.8–2.0 6/6 0.191–0.212
Protein isolates (100 mg) Nucleospin 118.2–136.1 1.7–2.0 7/7 0.24–23.6
Genespin 103.0–138.0 1.4–2.0 7/7 0.25–2.76
CTAB 20.6–42.6 1.7–2.0 7/7 0.041–8.52
Wizard 51.7–100.1 1.6–1.8 7/7 0.103–0.200
Soybean based drinks (300–500 mg) Nucleospin 0–0.35, 24.6* 2.0* 6/8 0.491*
Genespin 0–0.092, 13.8* 2.0* 6/8 0.276*
CTAB 0.285–7.60, 5.05* 1.4–2.0 8/8 0.070–6.72
Wizard 0.478–5.34, 6.47* 1.5–2.0 7/8 0.013–3.89
Soybean sauces (500 mg) Nucleospin 0–0.092 1.2–1.5 0/4 ND
Genespin 0.028–0.22 1.5–1.9 0/4 ND
CTAB 0–0.028 1.4–1.6 0/4 ND
Wizard 0–0.67 1.1–1.2 0/4 ND
Tofu (200 mg) Nucleospin 0.663–121.8 1.8–2.0 5/5 0.133–2.34
Genespin 0.109–105.9 1.6–1.8 5/5 0.057–21.2
CTAB 0.109–5.83 1.4–2.0 5/5 6.7  10 3–7.84
Wizard 10.2–103.8 1.7–1.9 5/5 0.020–0.280
Soybean desserts (500 mg) Nucleospin 0–20.8 1.0–2.0 3/6 0.089–0.417
Genespin 0–14.3 1.1–1.8 2/6 0.212–2.86
CTAB 0–5.28 1.5–2.0 3/6 2.2  10 4–5.9  10 3

Wizard 0.66–45.1 1.4–2.0 3/6 0.111–11.5

Vegetarian foods (200–300 mg) Nucleospin 0–64.7 1.0–1.8 2/6 1.29–32.4
Genespin 0–39.4 1.4–2.0 3/6 7.88–21.3
CTAB 0–14.0 1.1–1.9 4/6 8.81–27.7
Wizard 4.99–63.4 1.4–1.6 5/6 0.998–50.4
*
Values from the powder drink sample. ND, not detected.

of them it was observed a fragment above 10 kb (data

not shown).

3.2. PCR amplification of lectin gene

The results of PCR assays for DNA extracts of soybean

Fig. 2. Agarose gel electrophoresis of DNA extracted from soybean flour
using different methods of extraction. M, Hyperladder I (Bioline, London,
UK); lane 1, Nucleospin; lane 2, Genespin; lane 3, CTAB; lane 4, Wizard.
the four extraction methods. Fig. 2 shows an agarose gel
electrophoresis analysis of one sample of flour showing
prominent fragments above 10 kb, which suggests that
the DNA was not excessively sheared and was suitable
for amplification. Some degraded DNA and/or RNA con-
tamination could be observed with the kits and Wizard
methods, while no degraded DNA could be observed using
the CTAB method. All other processed food products ana-
lysed exhibit excessive DNA degradation because in none
flours, protein isolates and tofu show that the four methods
produced suitable DNA for PCR amplification, as all the
samples tested positively for the lectin gene presence (Table
1). For soybean flour, about 0.2 ng of extracted DNA were
enough to obtain a detectable product with a 35 cycle PCR.
For the protein isolates and tofu, the range of detection
limits was much higher than that for the soybean flours,
where the lowest levels of detection were obtained with
the CTAB method followed by the Wizard. The Nucleo-
Spin and GeneSpin kits show the lowest sensitivity for pro-
tein isolates and tofu, although this is compensated by the
highest DNA yields (Table 1).
The DNA extracts of soybean based drinks obtained
with the CTAB method were all successfully amplified
(Fig. 3C). Using the Wizard method one sample was con-
sidered not suitable for PCR amplification, as the product
obtained was practically not detectable (Fig. 3D). The kits
show good PCR amplification for the soybean powder
drink, but weak or non detectable fragments in all the other
1188 I. Mafra et al. / Food Control 19 (2008) 1183–1190
Fig. 3. Agarose gel electrophoresis of PCR products of soybean based
drink samples extracted with different methods. (A) Nucleospin;
(B) Genespin; (C) CTAB; (D) Wizard. M, 100 bp ladder (Bio-Rad,
Hercules, CA, USA); lane 1, soybean drink; lane 2, soybean drink; lane 3,
soybean and rice drink; lane 4, soybean drink; lane 5, soybean powder
drink; lane 6, soybean drink vanilla; lane 7, soybean drink; lane 8,
pineapple juice and soybean drink; lane B, negative control of extraction
reagents; lane C, negative control of PCR reagents.
drink samples (Fig. 3A, 3B), which is consistent with the
DNA yields obtained (Table 1). The lowest levels of detec-
tion were obtained with the Wizard method (Table 1).
The soybean sauces did not provide amplifiable DNA,
as in all the samples it was not possible to detect any
PCR product with the four extraction methods (Table 1).
The positive amplification of the spiked samples revealed
the absence of PCR inhibitors. The increased amount of
template DNA from 2 to 4 lL revealed the presence of
traces of PCR products in some samples extracted with
the kits and CTAB method. However, as the bands were
so slight, that was not considered a positive amplification
(data not shown).
The amplification of soybean desserts (Fig. 4, lanes 3–8)
was successfully achieved for three of the samples using the
CTAB (Fig. 4C), the NucleoSpin (Fig. 4A) and the Wizard
(Fig. 4D) methods. The best results were obtained using the
CTAB method, as it enabled the lowest levels of detection
(Table 1), which agrees with the higher band intensity
Fig. 4. Agarose gel electrophoresis of PCR products of soybean desserts
and vegetarian foods extracted with different methods. (A) Nucleospin;
(B) Genespin; (C) CTAB; (D) Wizard. M, 100 bp ladder (Bio-Rad,
Hercules, CA, USA); lane 1, soybean hamburger; lane 2, vegetable
hamburger with tofu; lane 3, soybean prepared for cream; lane 4,
strawberry soybean dessert; lane 5, strawberry soybean dessert; lane 6,
vanilla soybean dessert; lane 7, natural soybean dessert; lane 8, coffee and
hazelnut soybean ice-cream; lane 9, Croc tofu with tomato; lane 10,
Cordon Verde; lane 11, soybean sausage (apparently like Portuguese
smoked sausage); lane 12, soybean canned sausages; lane B, negative
control of extraction reagents; lane C, negative control of PCR reagents.
(Fig. 4C). The weak band obtained for sample 8 (lane)
using the Wizard method (Fig. 4D) was confirmed to be
positive by other replicate assays. The amplification of des-
sert extracts using the GeneSpin kit was only successful for
two of the samples (Fig. 4B).
The extracts obtained from vegetarian foods (Fig. 4,
lanes 1–2, 9–12) produced amplified products in four of
the samples by the CTAB (Fig. 4C) method and in five
samples by the Wizard (Fig. 4D) method. The undiluted
extracts for sample 9 (lane) gave positive amplification with
the CTAB and Wizard methods. The GeneSpin (Fig. 4B)
and NucleoSpin methods (Fig. 4A) kits enabled three and
two amplifications fragments, respectively, when applied
to vegetarian foods.
 I. Mafra et al. / Food Control 19 (2008) 1183–1190
Table 2
Coefficient of correlation and efficiency of real-time PCR standard curves
obtained from serially diluted DNA extracts from soybean flours
Extraction method Mean and SD*
R2 Slope E (%)**
Nucleospin 0.990 ± 0.005 3.327 ± 0.28 101.0 ± 12.7
Genespin 0.995 ± 0.003 3.062 ± 0.09 112.3 ± 4.7
CTAB 0.996 ± 0.003 3.212 ± 0.13 105.1 ± 5.8
Wizard 0.997 ± 0.003 3.063 ± 0.13 112.4 ± 7.0
*
SD, standard deviation.
**
E, Efficiency, E = [10( 1/slope)] 1.
3.3. Real-time PCR amplification of lectin gene
The real-time PCR assays were performed on the
extracts of soybean flours previously diluted to 10–50 ng/
lL. Each extract was serially diluted (1/2, 1/4, 1/8, 1/16,
1/32, 1/64 and 1/128) and amplified by real-time PCR with
SYBR Green I dye to yield standard curves. The linear cor-
relation coefficient of the standard curves (R2) ranged
between 0.980 and 0.999, and the PCR efficiency ranged
between 89.6% and 124.7% using the four extraction
methods, showing that all the DNA templates from the
different soybean flours were suitable for real-time PCR
quantification.
The ANOVA test was used to measure the significance
of differences among the real-time PCR efficiencies
obtained with the four methods (Table 2). This was possi-
ble because data obtained for each extraction presented
normal distribution (Shapiro–Wilk Test) and it was
observed homoscedasticity of variances (Levene Test).
The results of ANOVA test show that there are no signifi-
cant differences among the methods as P was higher than
0.05.
4. Discussion
The DNA isolation from food products is an important
achievement when considering the different types of indus-
trial processing that they have been subjected. The quality
of DNA is critical to obtain consistent, reliable and accu-
rate results, where the most important factors are the integ-
rity and purity.
In this study, the DNA from a range of food products
was extracted with four different methods. The samples
analysed included a range of soybean products, from the
very low processed such as the flours, to those highly pro-
cessed such as the sauces or vegetarian foods. To have reli-
able and wider results on each type of food matrix, the
samples were grouped (Table 1) for an easier comparison
of results. Each group of food products analysed included
samples from different suppliers and/or brands, which
means that each DNA isolation method was performed
many times in a wide range of products. The DNA isola-
tion methods tested in the present work were chosen from
those that have been successfully applied to other food
1189
matrices (Gryson et al., 2004; Olexová et al., 2004; Peano,
Samson, Palmierim, Gulli, & Marmiroli, 2004; Smith &
Maxwell, 2007).
The results show that all types of DNA isolation meth-
ods performed well for the majority of food products ana-
lysed, as evidenced by the amplification results. With the
exception of soybean sauces, some desserts and vegetarian
foods, the isolated DNA was present sufficient purity,
integrity and amount for PCR amplification. However,
some differences can be emphasised among the kits and
the other two methods, CTAB and Wizard.
As expected, the isolation of DNA from flours pro-
duced good quality extracts, as verified by other authors
(Abdullah, Radu, Hassan, & Hashim, 2006; Di Pinto
et al., 2007; Holden et al., 2003; Olexová et al., 2004).
However, the efficiencies of extraction varied with the
extraction protocol, as the highest yields were obtained
with the NucleoSpin and GeneSpin kits, whereas the
CTAB yielded the lowest DNA amounts. This finding
was also observed with the extraction of soybean defatted
flour and with wheat flour (Olexová et al., 2004), in oppo-
sition to the extraction of maize products whose results
evidenced the best performance for the Wizard method
(Peano et al., 2004; Smith & Maxwell, 2007). The kits pro-
vided also the best results for protein isolates and tofu,
meaning that when the samples are capable of producing
high DNA yields, the kits provided the best results. Con-
versely, for the samples more difficult to extract, such as
the drinks, the methods of choice are the CTAB or the
Wizard, as they provided the best results for DNA yield
and amplification. The other highly processed food prod-
ucts, namely, soybean desserts and vegetarian foods had
the best performance with the Wizard method for DNA
yield and similar amplification results with Wizard and
CTAB methods. These results agreed with other authors
who verified the optimal performance of CTAB method
in highly processed food matrices, when compared to
other commercial kits (Gryson et al., 2004). Furthermore,
other authors considered the Wizard method, generally,
the best for the extraction of DNA from potato-derived
foods, when compared to CTAB and several commercial
kits (Smith, Maxwell, & De Boer, 2005).
The soybean sauce is a product subjected to a severe
processing, which includes heat treatment, fermentation
in brine and refining. Thus, it is expected that the traces
of DNA found in the extracts should be much degraded
and very difficult to amplify. Kakihara, Matsufuji, Chino,
and Takeda (2006) evidenced the difficulties of obtaining
amplifiable DNA from fermented soybean products.
Because the extraction method can have a great influ-
ence on the results obtained by real-time PCR (Peano
et al., 2004), the amplification of the serially diluted
extracts obtained for soybean flours was performed by
real-time PCR. The comparison of the four extraction
methods showed that all the flour extracts presented similar
and good performances for real-time PCR quantification
as the efficiencies were close of 100% (Table 2).
1190 I. Mafra et al. / Food Control 19 (2008) 1183–1190
To conclude, the present work evidenced that the extrac-
tion of soybean flour or products with high DNA content
can be better achieved by the kits NucleoSpin and Gene-
Spin because both produced high purity and DNA yields,
with the advantage of being the fastest and simplest proto-
cols. The results show that those commercial kits should be
basically the same, so the choice should take in consider-
ation their cost. As the NucleoSpin kit is the less expensive
(3.1 €/sample compared to 6.3 €/sample for the GeneSpin,
both for 50 sample kit), this should be the method of choice
for soybean flours. When extracting difficult samples, the
best results for DNA quality and amount were obtained
with the CTAB and Wizard protocols. The CTAB method
presents the disadvantage of being time consuming com-
pared to the other methods, but it produces generally low
degraded and amplifiable DNA from most food samples.
The Wizard method presented a close performance to
CTAB, with the advantage of being simple, faster and eco-
nomic. Thus, the Wizard method can be a good choice for
highly processed soybean foodstuffs.
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